The invention provides novel polypeptides which are associated with the transcription complex NF-AT, polynucleotides encoding such polypeptides, antibodies which are reactive with such polypeptides, polynucleotide hybridization probes and PCR amplification probes for detecting polynucleotides which encode such polypeptides, transgenes which encode such polypeptides, homologous targeting constructs that encode such polypeptides and/or homologously integrate in or near endogenous genes encoding such polypeptides, nonhuman transgenic animals which comprise functionally disrupted endogenous genes that normally encode such polypeptides, and transgenic nonhuman animals which comprise transgenes encoding such polypeptides. The invention also provides methods for detecting T cells (including activated T cells) in a cellular sample, methods for treating hyperactive or hypoactive T cell conditions, methods for screening for immunomodulatory agents, methods for diagnostic staging of lymphocyte differentiation, methods for producing NF-AT proteins for use as research or diagnostic reagents, methods for producing antibodies reactive with the novel polypeptides, and methods for producing transgenic nonhuman animals.
The immune response is coordinated by the actions of cytokines produced from activated T lymphocytes, such as lymphocytes contacted with antigens. Cytokines are responsible for the control of proliferation and cell fate decisions among precursors of B cells, granulocytes and macrophages. T lymphocytes having a broad spectrum of antigen receptors are produced in the thymus as a product of the processes of selection and differentiation. When these T cells migrate to the peripheral lymphoid organs and encounter antigen, they undergo activation, during the process of which they produce large numbers of cytokines that act upon other cells of the immune system to coordinate their behavior to bring about an effective immune response.
T lymphocyte activation involves the specific regulation of many genes from minutes after the antigen encounter until at least 10 days later. T cells may also be activated by stimuli such as the combination of a calcium ionophore (e.g., ionomycin) and an activator of protein kinase C, such as phorbol myristate acetate (PMA). Several lectins, including phytohemagglutinin (PHA) may also be used to activate T cells (Nowell, P. C. (1990) Cancer Res. 20:462-466). The T cell activation genes are roughly grouped based on the time after stimulation at which each gene is regulated. Early genes trigger the regulation of subsequent genes in the activation pathway.
Because of the critical role of the T lymphocyte, agents that interfere with the early activation genes, such as cyclosporin A and FK506, are effective immunosuppressants. These early activation genes are regulated by transcription factors, such as NF-AT, that in turn are regulated through interactions with the antigen receptor. These transcription factors act through enhancer and promoter elements on the early activation genes to modulate their rate of transcription.
A typical early gene enhancer element is located in the first 325 base pairs upstream of the start site of the interleukin-2 gene. This region has been used extensively to dissect the requirements for T lymphocyte activation. This region binds an array of transcription factors including NF-AT, NFkB, Ap-1, Oct-1, and a newly identified protein that associates with Oct-1 called OAP-40. These different transcription factors act together to integrate the complex requirements for T lymphocyte activation.
The interieukin-2 gene is essential for both the proliferation and immunologic activation of T cells. The signaling pathways which connect the IL-2 gene and a representative and important early gene with the antigen receptor on the T cell surface and the signal transmission pathways between them are illustrated in FIG. 1. The binding site for the NF-AT protein appears to restrict expression of the interleukin-2 gene and other early activation genes to the context of an activated T lymphocyte. This information is based upon past work by Durand et al., Mol. and Cell. Biol. (1988), Shaw et al., Science, 241: 202 (1988), and Verwiej et al., (1990) J. Biol. Chem, 265: 15788-15795 (1990).
NF-AT appears to be the most important element among the group mentioned above in that it is able to direct transcription of any genes to activated T cells in the context of an intact transgenic animal (Verweij et al. J. Biol. Chem. 265:15788-15795, 1990). NF-AT is also the only element that requires physiologic activation through the antigen receptor for the activation of transcription by NF-AT. For example, the element is activated only after proper presentation of antigen of exactly the correct sequence by MHC-matched antigen presenting cells. This effect can be mimicked by pharmacologic agents, including the combination of ionomycin and PMA, which can also activate T cells through critical early genes.
Other elements within the IL-2 enhancer, for example, the NF-KB site or the AP-1 site, activate transcription in response to less specific stimuli, such as tumor necrosis factor alpha or simply PMA by itself These compounds do not activate the IL-2 gene and other early activation genes and do not lead to T cell activation. Such observations have led to the conclusion that NF-AT restricts the expression of certain early genes, such as the interleukin-2 gene to their proper biologic context. Preliminary data have also indicated that a selective genetic deficiency of NF-AT produces severe combined immunodeficiency (SCID) (Chatilla, T. et al. New Engl. J Med. 320:696-702, 1989).
As noted above, cyclosporin A (CsA) and FK506 are capable of acting as immunosuppressants. These agents inhibit T and B cell activation, mast cell degranulation and other processes essential to an effective immune response (Borel et al. (1976) Agents Actions 6: 468; Sung et al. (1988) J. Exp. Med. 168: 1539; Gao et al. Nature 336: 176). In T lymphocytes, these drugs disrupt an unknown step in the transmission of signals from the T cell antigen receptor to cytokine genes that coordinate the immune response (Crabtree et al. (1989) Science 243: 355; Schreiber et al. (1989) Science 251:283; Hohman and Hutlsch (1990) New Biol. 2: 663).
Putative intracellular receptors for FK506 and CsA have been described and found to be cis-trans prolyl isomerases (Fischer and Bang (1985) Biochim. Biophys. Acta 828: 39; Fischer et al. Nature 337: 476; Handschumacher et al. (1984) Science 226: 544; Lang and Schmid (1988) Nature 331: 453; Standaert et al. (1990) Nature 346: 671). Binding of the drugs inhibits isomerase activity; however, studies with other prolyl isomerase inhibitors (Bierer et al. (1990) Science 250: 556) and analysis of cyclosporin-resistant mutants in yeast suggest that the prevention of T lymphocyte activation results from formation of an inhibitory complex involving the drug and the isomerase (Bierer et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87: 9231; Tropschug et al. (1989) Nature 342: 953), and not from inhibition of the isomerase activity per se.
The transcription factor NF-AT appears to be a specific target of cyclosporin A and FK506, since transcription directed by this protein is completely blocked in T cells treated with these drugs, with little or no effect on other transcription factors, such as AP-1 and NF-KB (Shaw et al.(1988) op. cit; Emmel et al. (1989) Science 246: 1617; Mattila et al. (1990) EMBO J. 9: 4425). However, the drugs"" actual mechanism of action remains unclear. Unfortunately, while both are potent immunosuppressive agents, neither cyclosporin nor FK506 are ideal drugs.
For example, cyclosporin adverse reactions include renal dysfunction, tremors, nausea and hypertension. Indeed, for many years researchers have attempted to develop superior replacements, with FK506 being the most recent candidate. Without understanding how cyclosporin (or FK506) functions at the intracellular level, developing improved immunosuppressants represents an extremely difficult research effort with a very limited likelihood of success.
Thus, there exists a significant need to understand the functional basis of cyclosporin and FK506 effectiveness. With such knowledge, improved assays for screening drug candidates would become feasible, which could in turn dramatically enhance the search process. The present invention fulfills these and other needs.
The present invention provides novel methods and compositions useful, e.g., in screening for immunosuppressive agents. The invention is based in part on the discovery of the overall mechanism by which NF-AT is formed intracellularly from nuclear and cytoplasmic subunits and on the isolation of nucleic acids encoding NF-AT proteins.
A basis of the present invention is the discovery that NF-AT (i.e., a complex comprising NF-ATc and NF-ATn) is formed when a signal from the antigen receptor induces a preexisting cytoplasmic NF-AT submit (NF-ATc) to translocate to the nucleus and combine with a nuclear NF-AT subunit (NF-ATn). Cyclosporin A and FK506 block translocation of the cytoplasmic component without affecting the nuclear subunit. A plausible synthesis of these studies and previous work posits that the prolyl isomerases, FK506-binding protein (FK-BP) and cyclophilin, also function to import proteins to the nucleus.
The invention is also based on the purification of two related proteins, NF-ATc and NF-ATp, encoded by separate genes that represent the preexisting or cytosolic components of NF-AT. The carboxy-terminal half of NF-ATc shows limited similarity to the DNA binding and dimerization regions of the Dorsal/Rel family of transcription factors (FIG. 15, for review, Nolan and Baltimore (1992) Current Biology, Ltd. 2: 211-220) however, NF-ATc appears to be the most distantly related member of the group. Expression of a full length cDNA for one of these proteins, NF-ATc, activates the IL-2 promoter in non-T lymphocytes, while a dominant negative of NF-ATc specifically blocks activation of the IL-2 promoter in T lymphocytes, indicating that NF-ATc is required for IL-2 gene expression and is responsible for the restricted expression of IL-2. NF-ATc RNA expression is largely restricted to lymphoid tissues and is induced upon cell activation. The second protein, NF-ATp, is highly homologous to NF-ATc over a limited domain, but exhibits wider tissue distribution and is highly expressed in tissues characterized by Ca++-dependent regulation. Together these proteins are members of a new family of DNA binding proteins, which are distantly related to the Dorsal/Rel family (Nolan and Baltimore (1992) Current Biology, Ltd. 2: 211-220). Agents that increase intracellular Ca++ or that activate protein kinase C independently produce alterations in the mobility of NF-ATc, indicating that distinct signaling pathways converge on NF-ATc to regulate its function.
In accordance with one aspect of the invention, novel compositions include NF-ATc polypeptides, nuclear components of NF-AT complexes, e.g, an NF-ATn polypeptide, mixtures of the polypeptides, and cellular extracts containing the polypeptides. The NF-ATn and NF-ATc subunits are capable of forming a native NF-AT complex which binds in a sequence-specific manner to a transcriptional regulatory DNA sequence of an immune response gene. The NF-ATn subunit is characterized by:
i. a molecular weight of about 45 kd;
ii. inducible expression in T cells (such as Jurkat cells);
iii. inducible expression in HeLa cells by exposing the cells to an agent (such as PMA) capable of activating protein kinase C;
iv. a lack of effect by cyclosporin and FK506 on NF-ATn synthesis in T cells; and
v. specifically binding to an NF-ATc.
The NF-ATc subunit is characterized by:
i. a molecular weight of about 90 kd;
ii. constitutively expressed in T cells;
iii. ability to be transported into a nucleus after a Ca++ flux in the cell;
iv. nuclear transport inhibited by cyclosporin and FK506; and
v. specifically binding to an NF-ATn.
In another aspect of the present invention, isolated or purified nucleic acid sequences (or their complementary sequences) are provided which are capable of binding to an NFAT complex, wherein the sequences are substantially homologous to an enhancer, such as IL-2 and IL-4 enhancers, particularly the sequence AAGAGGAAAAA (SEQ ID NO: 53).
In another aspect, the invention embraces methods of screening for an immune regulating agent comprising combining the agent with a component selected from the group consisting of an NF-ATn polypeptide, an NF-ATc polypeptide, and mixtures thereof; and determining whether the agent binds to the selected component.
In another embodiment, candidate immunomodulatory agents are identified by their ability to block the binding of a NF-ATc polypeptide to other components of NF-AT (e.g., AP-1) and/or to block the binding of NF-AT to DNA having an NF-AT recognition site. The DNA preferably includes one or more NF-AT binding sites at which a NF-AT protein complex specifically binds. One means for detecting binding of a NF-AT protein comprising NF-ATc to DNA is to immobilize the DNA, such as by covalent or noncovalent chemical linkage to a solid support, and to contact the immobilized DNA with a NF-AT protein complex comprising a NF-ATc polypeptide that has been labeled with a detectable marker (e.g., by incorporation of radiolabeled amino acid). Such contacting is typically performed in aqueous conditions which permit binding of a NF-AT protein to a target DNA containing a NF-AT binding sequence. Binding of the labeled NF-AT to the immobilized DNA is measured by determining the extent to which the labeled NF-ATc polypeptide is immobilized as a result of a specific binding interaction. Such specific binding may be reversible, or may be optionally irreversible if a cross-linking agent is added in appropriate experimental conditions.
In yet another embodiment, methods of screening for an immune regulating agent will comprise the steps of:
i. preparing a collection of eukaroytic cells containing NF-ATc in the cytoplasm of the cell;
ii. treating the cells with an agent;
iii. assaying for nuclear translocation of the NF-ATc wherein blocking of nuclear transport correlates with the immunosuppressive activity of the agent. The step of assaying for nuclear translocation preferably comprises determining the nuclear presence of the NF-ATc which is labeled with a detectable marker. Alternatively, the step of assaying for nuclear translocation comprises determining nuclear association between the NF-ATc and an NF-ATn, preferably using nuclei treated previously with the agent.
The assaying step can also comprise determining binding of NF-AT to a DNA sequence in the cell, such as by determining mRNA transcription levels in the cell, wherein the mRNA encodes an immune response gene.
In a different embodiment, the method of screening for immune regulating agents can comprise:
i. constructing a chimeric gene comprising an NF-AT regulated enhancer region linked to a reporter gene (e.g., chloramphenicol acetyltransterase (CAT) gene);
ii. inserting the chimeric gene into T cells;
iii. treating the T cells with T cell activating compounds in the presence or absence of the agent; and
iv. determining the effect of the agent on expression of the reporter gene. Inhibition of expression of the reporter gene indicates that the agent is a candidate immunosuppressant agent.
In one aspect, candidate immunomodulatory agents are identified as being agents capable of inhibiting (or enhancing) intermolecular binding between NF-ATc and other polypeptides which comprises a NF-AT complex (e.g., AP-1, JunB, etc.). The invention provides methods and compositions for screening libraires of agents for the capacity to interfere with binding of NF-ATc to other NF-AT polypeptide species under aqueous binding conditions. Typically, at least either NF-ATc and/or another NF-AT polypeptide species is labeled with a detectable label and intermolecular binding between NF-ATc and other NF-AT polypeptide species is detected by the amount of labeled species captured in NF-AT complexes and the like.
For example, methods of assaying for a candidate-immunosuppressant agent comprise mixing the agent with NF-ATn and NF-ATc under conditions which permit specific multimerization to form NF-AT, comprising dimerization of NF-ATn and NF-ATc, and determining whether said dimerization (and/or multimerization with other subunits) occurs. Typically, NF-ATn or NF-ATc is immobilized and at least one subunit is labeled with a detectable marker, most usually the non-immobilized NF-AT subunit is labeled.
The present invention further provides several novel methods and compositions for modulating the immune response and for screening for modulators of the immune response which utilize polynucleotide sequences encoding NF-ATc recombinant proteins and complementary polynucleotides which are substantially identical to NF-ATc polynucleotide sequences.
Thus, in another aspect of the invention, NF-ATc polypeptides comprising polypeptide sequences which are substantially identical to a sequence shown in FIG. 12 or a cognate NF-ATc amino acid sequence are provided.
The invention also provides for nucleic acid sequences encoding NF-ATc. The characteristics of the cloned sequences are given, including the nucleotide and predicted amino acid sequence in FIG. 12. Polynucleotides comprising these sequences can serve as templates for the recombinant expression of quantities of NF-ATc polypeptides, such as human NF-ATc and murine NF-ATc. Polynucleotides comprising these sequences can also serve as probes for nucleic acid hybridization to detect the transcription and mRNA abundance of NF-ATc mRNA in individual lymphocytes (or other cell types) by in situ hybridization, and in specific lymphocyte populations by Northern blot analysis and/or by in situ hybridization (Alwine et al. (1977) Proc. Natl. Acad. Sci. U.S.A. 74: 5350) and/or PCR amplification and/or LCR detection. Such recombinant polypeptides and nucleic acid hybridization probes have utility for in vitro screening methods for immunomodulatory agents and for diagnosis and treatment of pathological conditions and genetic diseases, such as transplant rejection reactions, T cell-mediated immune responses, lymphocytic leukemias (e.g., T cell leukemia or lymphoma) wherein NF-AT activity contributes to disease processes, autoimmune disease, arthritis, and the like.
The invention also provides antisense polynucleotides complementary to NF-ATc sequences which are employed to inhibit transcription and/or translation of the cognate mRNA species and thereby effect a reduction in the amount of the respective NF-ATc protein in a cell (e.g., a T lymphocyte of a patient). Such antisense polynucleotides can function as immunomodulatory drugs by inhibiting the formation of NF-AT protein required for T cell activation.
In a variation of the invention, polynucleotides of the invention are employed for diagnosis of pathological conditions or genetic disease that involve T cell neoplasms or T cell hyperfunction or hypofunction, and more specifically conditions and diseases that involve alterations in the structure or abundance of NF-ATc polypeptide, NF-ATc polynucleotide sequence, or structure of the NF-ATc gene or flanking region(s).
The invention also provides antibodies which bind to NF-ATc with an affinity of about at least 1xc3x97107 Mxe2x88x921 and which lack specific high affinity binding for other proteins present in activated T cells. Such antibodies can be used as diagnostic reagents to identify T cells (e.g., activatable T cells) in a cellular sample from a patient (e.g., a lymphocyte sample, a solid tissue biopsy) as being cells which contain an increased amount of NF-ATc protein determined by standardization of the assay to be diagnostic for activated T cells. Frequently, anti-NF-ATc c antibodies are included as diagnostic reagents for immunohistopathology staining of cellular samples in situ. Additionally, anti-NF-ATc antibodies may be used therapeutically by targeted delivery to T cells (e.g., by cationization or by liposome/immunoliposome delivery).
The invention also provides NF-ATc polynucleotide probes for diagnosis of neoplasia or immune status by detection of NF-ATc mRNA in cells explanted from a patient, or detection of a pathognomonic NF-ATc allele (e.g., by RFLP or allele-specific PCR analysis). A pathognomonic NF-ATc allele is an allele which is statistically correlated with the presence of a predetermined disease or propensity to develop a disease. Typically, the detection will be by in situ hybridization using a labeled (e.g., 32P, 35S, 14C, 3H, fluorescent, biotinylated, digoxigeninylated) NF-ATc polynucleotide, although Northern blotting, dot blotting, or solution hybridization on bulk RNA or poly A+ RNA isolated from a cell sample may be used, as may PCR amplification using NF-ATc-specific primers. Cells which contain an increased amount of NF-ATc mRNA as compared to standard control values for cells or cell types other than activated T cells or activatable T cells will be thereby identified as activated T cells or activatable T cells. Similarly, the detection of pathognomonic rearrangements or amplification of the NF-ATc locus or closely linked loci in a cell sample will identify the presence of a pathological condition or a predisposition to developing a pathological condition (e.g., cancer, genetic disease).
The present invention also provides a method for diagnosing T cell hypofunction or hyperfunction in a human patient, wherein a diagnostic assay (e.g., immunohistochemical staining of fixed lymphocytic cells by an antibody that specifically binds human NF-ATc) is used to determine if a predetermined pathognomonic concentration of NF-ATc protein or NF-ATc mRNA is present in a biological sample from a human patient; if the assay indicates the presence of NF-ATc protein or NF-ATc mRNA at or above such predetermined pathognomonic concentration, the patient is diagnosed as having T cell hyperfunction or hypofunction condition, or transplant rejection and the like.
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.